A cascade involving a series of enzymes and called prophenoloxidase activating system (hereinafter referred to as proPO activating system), which is concerned with melanin formation in body fluid, exists in the body fluid of insects and .beta.-1,3-glucan, a fungal cell wall component, and peptidoglycan, a bacterial cell wall component, are known to trigger said cascade [Onishi, Annot. Zool. Jpn., 27, 33-39 (1954), Ashida and Onishi, Arch. Biochem. Biophys., 122, 411-416 (1967), Brunet, Insect Biochem., 10, 467-500 (1980); Ashida and Yamazaki, Molting and Metamorphosis, 239-265, Japan Sci. Soc. Press (1990)]. This action is ascribed to .beta.-1,3-glucan recognition protein (BGRP; molecular weight 62 kD) exhibiting specific affinity to .beta.-1,3-glucan and peptidoglycan recognition protein (PGRP; molecular weight 19 kD) exhibiting specific affinity to peptidoglycan, which are present in said proPO activating system. When .beta.-1,3-glucan or peptidoglycan is recognized by either protein, a series of cascade reactions begins. Through some unelucidated reactions inclusive of the reaction of an enzyme requiring Ca.sup.2+, a PPAE which converts prophenoloxidase (precursor of phenoloxidase, hereinafter referred to as proPO) to phenoloxidase (hereinafter referred to as PO) is produced [Ashida and Dohke, Insect Biochem., 10, 37-47 (1980)].
In view of such phenomenon, various studies have been done in an attempt to establish a method for detecting bacterial infections and fungal infections, and safety tests of therapeutic agents by assaying .beta.-1,3-glucan and peptidoglycan based on said cascade reaction.
Currently, Limulus test reagents, using amebocyte lysate from horseshoe crab (Limulus polyphemus, Tachypleus tridentatus) also having an in vivo cascade similar to that of insects, have been used as reagents for detecting bacterial infections and safety tests of therapeutic agents etc. In the absence of a system which reacts with peptidoglycan in horseshoe crab, however, detection is possible only for .beta.-1,3-glucan and lipopolysaccharides, excluding Gram positive bacteria having no lipopolysaccharides. In addition, the reagent supplier horseshoe crab (Tachypleus tridentatus) is limited in number in Japan and unstable future supply thereof will always pose a difficulty.
When the cascade (proPO activating system) of insects is utilized, on the other hand, stable supply of reagents, as well as detection of bacteria, not to mention fungi, inclusive of Gram positive and negative ones can be expected, since the cascade includes a system which responds to peptidoglycan. It is also possible to specifically assay .beta.-1,3-glucan (fungus) and peptidoglycan (bacterium) independently by separating a component which specifically reacts with peptidoglycan or a component which specifically reacts with .beta.-1,3-glucan from the body fluid (U.S. Pat. No. 4,970,152).
The assay method based on the cascade of insects includes a conventional method wherein .beta.-1,3-glucan and peptidoglycan are detected by measuring PO activity developed upon conversion of proPO to PO by PPAE. This method is encountered with the problems of self-oxidation of PO (which is an unstable enzyme) and unattainable markedly enhanced detection sensitivity.
So as to solve such problems, Kenneth T. Soderhall proposes a method for measuring .beta.-1,3-glucan and endotoxin (lipopolysaccharides) by assaying certain serine protease activity which activates PO, with the use of a synthetic peptide as a substrate (EP-A-96689). The substrate used in this method is constructed without considering the reaction specificity of PPAE, which catalyzes the conversion of proPO to PO, but based on the sole fact that PPAE is among serine proteases, and whether PPAE activity, let alone .beta.-1,3-glucan and endotoxin (lipopolysaccharides), can be determined with precision is moot.